Intra-epithelial non-canonical Activin A signaling safeguards prostate progenitor quiescence.

The healthy prostate is a relatively quiescent tissue. Yet, prostate epithelium overgrowth is a common condition during aging, associated with urinary dysfunction and tumorigenesis. For over thirty years, TGF-ß ligands have been known to induce cytostasis in a variety of epithelia, but the intracellular pathway mediating this signal in the prostate, and its relevance for quiescence, have remained elusive. Here, using mouse prostate organoids to model epithelial progenitors, we find that intra-epithelial non-canonical Activin A signaling inhibits cell proliferation in a Smad-independent manner. Mechanistically, Activin A triggers Tak1 and p38 ΜAPK activity, leading to p16 and p21 nuclear import. Spontaneous evasion from this quiescent state occurs upon prolonged culture, due to reduced Activin A secretion, a condition associated with DNA replication stress and aneuploidy. Organoids capable to escape quiescence in vitro are also able to implant with increased frequency into immunocompetent mice. This study demonstrates that non-canonical Activin A signaling safeguards epithelial quiescence in the healthy prostate, with potential implications for the understanding of cancer initiation, and the development of therapies targeting quiescent tumor progenitors.

AGO2 promotes telomerase activity and interaction between the telomerase components TERT and TERC.

Telomerase reverse transcriptase (TERT) and telomerase RNA component (TERC) constitute the core telomerase enzyme that maintains the length of telomeres. Telomere maintenance is affected in a broad range of cancer and degenerative disorders. Taking advantage of gain- and loss-of-function approaches, we show that Argonaute 2 (AGO2) promotes telomerase activity and stimulates the association between TERT and TERC AGO2 depletion results in shorter telomeres as well as in lower proliferation rates in vitro and in vivo We also demonstrate that AGO2 interacts with TERC and with a newly identified sRNA (terc-sRNA), arising from the H/ACA box of TERC Notably, terc-sRNA is sufficient to enhance telomerase activity when overexpressed. Analyses of sRNA-Seq datasets show that terc-sRNA is detected in primary human tissues and increases in tumors as compared to control tissues. Collectively, these data uncover a new layer of complexity in the regulation of telomerase activity by AGO2 and might lay the foundation for new therapeutic targets in tumors and telomere diseases.

Effects of the Combined Treatment with a G-Quadruplex-Stabilizing Ligand and Photon Beams on Glioblastoma Stem-like Cells: A Magnetic Resonance Study.

Glioblastoma multiforme is a malignant primary brain tumor with a poor prognosis and high rates of chemo-radiotherapy failure, mainly due to a small cell fraction with stem-like properties (GSCs). The mechanisms underlying GSC response to radiation need to be elucidated to enhance sensitivity to treatments and to develop new therapeutic strategies. In a previous study, two GSC lines, named line #1 and line #83, responded differently to carbon ions and photon beams, with the differences likely attributable to their own different metabolic fingerprint rather than to radiation type. Data from the literature showed the capability of RHPS4, a G-quadruplex stabilizing ligand, to sensitize the glioblastoma radioresistant U251MG cells to X-rays. The combined metabolic effect of ligand #190, a new RHPS4-derivative showing reduced cardiotoxicity, and a photon beam has been monitored by magnetic resonance (MR) spectroscopy for the two GSC lines, #1 and #83, to reveal whether a synergistic response occurs. MR spectra from both lines were affected by single and combined treatments, but the variations of the analysed metabolites were statistically significant mainly in line #1, without synergistic effects due to combination. The multivariate analysis of ten metabolites shows a separation between control and treated samples in line #1 regardless of treatment type, while separation was not detected in line #83.

Inhibition of Bromodomain and Extraterminal Domain (BET) Proteins by JQ1 Unravels a Novel Epigenetic Modulation to Control Lipid Homeostasis.

The homeostatic control of lipid metabolism is essential for many fundamental physiological processes. A deep understanding of its regulatory mechanisms is pivotal to unravel prospective physiopathological factors and to identify novel molecular targets that could be employed to design promising therapies in the management of lipid disorders. Here, we investigated the role of bromodomain and extraterminal domain (BET) proteins in the regulation of lipid metabolism. To reach this aim, we used a loss-of-function approach by treating HepG2 cells with JQ1, a powerful and selective BET inhibitor. The main results demonstrated that BET inhibition by JQ1 efficiently decreases intracellular lipid content, determining a significant modulation of proteins involved in lipid biosynthesis, uptake and intracellular trafficking. Importantly, the capability of BET inhibition to slow down cell proliferation is dependent on the modulation of cholesterol metabolism. Taken together, these data highlight a novel epigenetic mechanism involved in the regulation of lipid homeostasis.

Robotic versus laparoscopic radical nephrectomy: a large multi-institutional analysis (ROSULA Collaborative Group).

OBJECTIVE: To compare the outcomes of robotic radical nephrectomy (RRN) to those of laparoscopic radical nephrectomy (LRN) for large renal masses. METHODS: This was a retrospective analysis of RRN and LRN cases performed for large (≥ cT2) renal masses from 2004 to 2017 and collected in the multi-institutional international database (ROSULA: RObotic SUrgery for LArge renal masses). Peri-operative, functional, and oncologic outcomes were compared between each approach. Descriptive analyses were performed and presented as medians with interquartile ranges. Inverse probability of treatment weighting-adjusted multivariable analyses were used to identify predictors of peri-operative complications. Kaplan-Meier analysis and Cox regression models were used to assess survival outcomes. RESULTS: A total of 941 patients (RRN = 404, LRN = 537) were identified. There was no difference in terms of gender, age, and clinical tumor size. Over the study period, RRN had an annual increase of 11.75% (95% CI [7.34, 17.01] p < 0.001) and LRN had an annual decline of 5.39% (95% CI [-6.94, -3.86] p < 0.001). Patients undergoing RRN had higher BMI (27.6 [IQR 24.8-31.1] vs. 26.5 [24.1-30.0] kg/m2, p < 0.01). Operative duration was longer for RRN (185.0 [150.0-237.2] vs. 126 [90.8-180.0] min, p < 0.001). Length of stay was shorter for RRN (3.0 [2.0-4.0] vs. 5.0 [4.0-7.0] days, p < 0.001). RRN cases presented more advanced disease (higher pathologic staging [pT3-4 52.5 vs. 24.2%, p < 0.001], histologic grade [high grade 49.3 vs. 30.4%, p < 0.001], and rate of nodal disease [pN1 5.4 vs. 1.9%, p < 0.01]). Surgical approach did not represent an independent risk factor for peri-operative complications (OR 1.81 95% CI [0.97-3.39], adjusted p = 0.2). The main study limitation is the retrospective design. CONCLUSIONS: This study represents the largest known multi-center comparison between RRN and LRN. The two procedures seem to offer similar peri-operative outcomes. Notably, RRN has been increasingly utilized, especially in the setting of more advanced and surgically challenging disease without increasing the risk of peri-operative complications.

Physiological and pathological levels of prostaglandin E2 in renal parenchyma and neoplastic renal tissue.

Prostaglandin (PG)E2 seems to promote tumor proliferation by regulating cell growth, inhibiting apoptosis, promoting angiogenesis, and suppressing host immune surveillance of cancer cells. The suppression of prostaglandins biosynthesis is thought to be the main molecular mechanism for non-steroidal anti-inflammatory drugs antineoplastic effect. Yet the relationship between PGE2 and human renal cell carcinoma remains unclear. The aim of our study is to evaluate the PGE2 content in human renal parenchyma and Renal Cell Carcinoma. The study was conducted on 20 consecutive patients undergoing radical nephrectomy for Renal Cell Carcinoma. In the normal renal parenchyma and in the neoplastic renal tissue the PGE2 level was 83.43 ± 5.89 pg/mg and 289.67 ± 22.2 pg/mg, respectively (P < 0.0001). There was no relationship between PGE2 content and Renal Cell Carcinoma dimension, Fuhrman grade, pathological-Tumor-Node and Metastasis (pTNM) stage and histological subtype. The PGE2 over-content in neoplastic renal tissue suggests a role of PGE2 in development and progression of renal carcinoma.

Telomere Length Maintenance in Cancer: At the Crossroad between Telomerase and Alternative Lengthening of Telomeres (ALT).

Eukaryotic cells undergo continuous telomere shortening as a consequence of multiple rounds of replications. During tumorigenesis, cells have to acquire telomere DNA maintenance mechanisms (TMMs) in order to counteract telomere shortening, to preserve telomeres from DNA damage repair systems and to avoid telomere-mediated senescence and/or apoptosis. For this reason, telomere maintenance is an essential step in cancer progression. Most human tumors maintain their telomeres expressing telomerase, whereas a lower but significant proportion activates the alternative lengthening of telomeres (ALT) pathway. However, evidence about the coexistence of ALT and telomerase has been found both in vivo in the same cancer populations and in vitro in engineered cellular models, making the distinction between telomerase- and ALT-positive tumors elusive. Indeed, after the development of drugs able to target telomerase, the capability for some cancer cells to escape death, switching from telomerase to ALT, was highlighted. Unfortunately, to date, the mechanism underlying the possible switching or the coexistence of telomerase and ALT within the same cell or populations is not completely understood and different factors could be involved. In recent years, different studies have tried to shed light on the complex regulation network that controls the transition between the two TMMs, suggesting a role for embryonic cancer origin, epigenetic modifications, and specific genes activation-both in vivo and in vitro. In this review, we examine recent findings about the cancer-associated differential activation of the two known TMMs and the possible factors implicated in this process. Furthermore, some studies on cancers are also described that did not display any TMM.

Impact of novel techniques on minimally invasive adrenal surgery: trends and outcomes from a contemporary international large series in urology.

OBJECTIVE: To evaluate contemporary international trends in the implementation of minimally invasive adrenalectomy and to assess contemporary outcomes of different minimally invasive techniques performed at urologic centers worldwide. METHODS: A retrospective multinational multicenter study of patients who underwent minimally invasive adrenalectomy from 2008 to 2013 at 14 urology institutions worldwide was included in the analysis. Cases were categorized based on the minimally invasive adrenalectomy technique: conventional laparoscopy (CL), robot-assisted laparoscopy (RAL), laparoendoscopic single-site surgery (LESS), and mini-laparoscopy (ML). The rates of the four treatment modalities were determined according to the year of surgery, and a regression analysis was performed for trends in all surgical modalities. RESULTS: Overall, a total of 737 adrenalectomies were performed across participating institutions and included in this analysis: 337 CL (46 % of cases), 57 ML (8 %), 263 LESS (36 %), and 80 RA (11 %). Overall, 204 (28 %) operations were performed with a retroperitoneal approach. The overall number of adrenalectomies increased from 2008 to 2013 (p = 0.05). A transperitoneal approach was preferred in all but the ML group (p < 0.001). European centers mostly adopted CL and ML techniques, whereas those from Asia and South America reported the highest rate in LESS procedures, and RAL was adopted to larger extent in the USA. LESS had the fastest increase in utilization at 6 %/year. The rate of RAL procedures increased at slower rates (2.2 %/year), similar to ML (1.7 %/year). Limitations of this study are the retrospective design and the lack of a cost analysis. CONCLUSIONS: Several minimally invasive surgical techniques for the management of adrenal masses are successfully implemented in urology institutions worldwide. CL and LESS seem to represent the most commonly adopted techniques, whereas ML and RAL are growing at a slower rate. All the MIS techniques can be safely and effectively performed for a variety of adrenal disease.

A prospective multicenter European study on flexible ureterorenoscopy for the management of renal stone.

PURPOSE: The aim of this study was to describe the outcomes and the complications of retrograde intrarenal surgery (RIRS) for renal stones in a multi-institutional working group. MATERIALS AND METHODS: From 2012 to 2014, we conducted a prospective study including all RIRS performed for kidney stones in 4 European centers. Demographic information, disease characteristics, and perioperative and postoperative data were gathered. Patients and stone data, procedure characteristics, results and safety outcomes were analyzed and compared by descriptive statistics. Complications were reported using the standardized Clavien system. RESULTS: Three hundred and fifty-six patients underwent 377 RIRS with holmium laser lithotripsy for renal stones. The RIRS was completed in all patients with a mean operative time of 63.5 min. The stone-free status was confirmed endoscopically and through fluoroscopic imaging after the first procedure in 73.6%. The second procedure was performed in twenty patients (5.6%) achieving an overall stone free rate of 78.9%. The overall complication rate was 15.1%. Intra-operative and post-operative complications were seen in 24 (6.7%) and 30 (8.4%) cases, respectively. CONCLUSIONS: RIRS is a minimally invasive procedure with good results in terms of stone-free and complications rate.

Intraoperative presentation of Bochdalek's hernia in an adult during robotic-assisted partial nephrectomy: An uncommon situation and literature review.

Bochdalek's diaphragmatic hernia (BDH) is a congenital defect of the diaphragm that usually present during the neonatal period and rarely remain silent until adulthood. We present a 45-year-old-female case with diagnosis of double left kidney tumor prepared for robot-assisted partial nephrectomy (RPN). During the preoperative procedure she had a reduction of inspiratory volumes and increased pulmonary pressures: the robotic camera revealed the incidental presence of the left diaphragmatic defect. We report a simultaneous nephron sparing surgery (NSS) and left posterolateral BDH correction done by the da Vinci Surgical Robot (Intuitive Surgical, Sunnyvale, CA).

Giant renal artery aneurysm: A case report.

A case of a 12 cm giant renal artery aneurysm (RAA) in an 59-year-old woman is reported. The patient was referred to our hospital for flank pain and spot hematuria. Ultrasonography (US) revealed some wide lacunar areas in her right kidney and a thin cortex. Three-dimensional computed tomography (3D-CT) revealed a giant right renal arteriovenous malformation (AVM). AngioCT scan showed a pervious right renal artery. The cavities of the right kidney were dilated and the parenchyma was markedly reduced. Two months later the patient underwent an open resection of the aneurysm and a right nephrectomy. She had an uneventful recovery and a healthy status (last follow-up: 9 month). In this particular case, a safe approach is the transabdominal approach since the aneurysm was very large, friable, and located on the right side. This report confirms the opportunity of a planned nephrectomy once there is adequate renal reserve in the opposite kidney using a midline approach.

Study of genotoxic and cytotoxic effects induced in human fibroblasts by exposure to pulsed and continuous 1.6 GHz radiofrequency.

Background: The widespread use of radiofrequency (RF) sources, ranging from household appliances to telecommunications devices and military equipment, raises concerns among people and regulatory agencies about the potential health risks of RF exposure. Consequently, several in vitro and in vivo studies have been done to investigate the biological effects, in particular non-thermal, of this non-ionizing radiation. To date, this issue is still being debated due to the controversial results that have been reported. Furthermore, the impact of different RF signal modulations on biological systems remains poorly investigated. The present in vitro study aims to evaluate the cytotoxicity and genotoxicity of continuous or pulsed 1.6 GHz RF in human dermal fibroblasts (HDF). Methods: HDF cultures were exposed to continuous and pulsed 1.6 GHz RF, for 2 h, with Specific Absorption Rate (SAR) of 0.4 W/kg. The potential biological effects of 1.6 GHz RF on HDF were assessed with a multi-methodological approach, analyzing the effects on cell cycle, ultrastructure, protein expression, mitotic spindle, CREST stained micronuclei, chromosome segregation and γ-H2AX/53BP1 foci. Results: 1.6 GHz RF exposure modified proteins expression and morphology of HDF. Specifically, the expression of different heat-shock proteins (HSP) (i.e., HSP-90, HSP-60, and HSP-25) and phospho-AKT were affected. In addition, both continuous and pulsed RF modified the cytoskeletal organization in HDF and increased the number of lysosomes, while the formation of autophagosomes was observed only after pulsed RF exposure. Mitotic spindle anomalies were also found after exposure. However, no significant effect was observed on cell cycle, chromosome segregation, CREST-stained micronuclei and γ-H2AX/53BP1 foci. Conclusion: The results of the present study show the absence of genotoxic damage in 1.6 GHz RF exposed HDF and, although mitotic spindle alterations were observed, they did not have an aneugenic effect. On the other hand, changes in some proteins expression and cell ultrastructure in exposed HDF suggest that RF can potentially induce cell alterations at the morphological and molecular levels.

Assessing the accuracy and generalizability of the preoperative and postoperative Karakiewicz nomograms for renal cell carcinoma: results from a multicentre European and US study.

OBJECTIVE: To assess the accuracy and generalizability of the pre- and postoperative Karakiewicz nomograms for predicting cancer-specific survival (CSS) in patients with renal cell carcinoma (RCC). PATIENTS AND METHODS: This retrospective study included 3231 patients from European and US centres, who were treated by radical or partial nephrectomy for RCC between 1992 and 2010. Prognostic scores for each patient were calculated and the primary endpoint was CSS. Discriminating ability was assessed by Harrell's c-index for censored data. The 'validation by calibration' method proposed by Van Houwelingen was used for checking the calibration of covariate effects. Calibration was graphically explored. RESULTS: Local and systemic symptoms were present in 23.2% and 9.1% of the patients, respectively. The median follow-up (FU) was 49 months. At the last FU, 408 cancer-related deaths were recorded, Kaplan-Meier estimates of CSS (with 95% confidence intervals [CIs]) at 5 and 10 years were 0.86 (0.84-0.87) and 0.77 (0.75-0.80), respectively. Both nomograms discriminated well. Stratified c-indices for CSS were 0.784 (95% CI 0.753-0.814) for the preoperative nomogram, and 0.842 (95% CI 0.816-0.867) for the postoperative one, with a significant difference between the two values (P < 0.001). The covariate-based predictions on our data for both nomograms were valid. The calibration plots showed no relevant departures from ideal predictions. CONCLUSIONS: The results suggest that the postoperative Karakiewicz nomogram discriminates substantially better than the preoperative one. These nomogram-based predictions may be used as benchmark data for pretreatment and postoperative decision-making in patients at various stages of RCC.

The effects of dutasteride and finasteride on BPH-related hospitalization, surgery and prostate cancer diagnosis: a record-linkage analysis.

PURPOSE: To investigate differences in the risk of benign prostatic hyperplasia (BPH)-related hospitalization, for surgical and non-surgical reasons, and of new prostate cancer (PCa) diagnosis between patients using finasteride or dutasteride. METHODS: A retrospective cohort study was conducted using data from record linkage of administrative databases (pharmaceutical prescription data, hospital discharge records, Italian population registry). Men aged ≥ 40 years old who had received a prescription for at least 10 packs/year between January 1, 2004 and December 31, 2004 were included and followed for 5 years. The association of the outcomes was assessed using a multiple Cox proportional hazard model. Propensity score-matched analysis and a 5-1, greedy 1:1 matching algorithm were performed. RESULTS: 8,132 patients were identified. Overall incidence rates of BPH hospitalization and BPH-related surgery were 21.05 (95 % CI 19.52-22.71) and 20.97 (95 % CI 19.45-22.61) per 1,000 person-years, respectively. In the dutasteride group compared with finasteride group, the incidence rate of both events was statistically significant lower: 16.07 versus 21.76 for BPH hospitalization and 15.91 versus 21.69 for BPH-related surgery. The incidence rate of new PCa was also lower for the dutasteride group [8.34 (95 % CI 5.96-11.68) vs. 10.25 (95 % CI 9.15-11.49)]. Dutasteride was associated with a reduction in BPH-related hospitalizations (HR 0.75, 95 % CI 0.58-0.98 and 0.58-0.98 for surgical and non-surgical reasons). The matched analysis confirmed the risk reduction with dutasteride for BPH-related surgery. CONCLUSIONS: These findings suggest that the clinical effects of dutasteride and finasteride might be different. Patients treated with dutasteride seem to be less likely to experience BPH-related hospitalization. Comparative studies are needed to confirm these results.

NBN Gene Polymorphisms and Cancer Susceptibility: A Systemic Review.

The relationship between DNA repair failure and cancer is well established as in the case of rare, high penetrant genes in high cancer risk families. Beside this, in the last two decades, several studies have investigated a possible association between low penetrant polymorphic variants in genes devoted to DNA repair pathways and risk for developing cancer. This relationship would be also supported by the observation that DNA repair processes may be modulated by sequence variants in DNA repair genes, leading to susceptibility to environmental carcinogens. In this framework, the aim of this review is to provide the reader with the state of the art on the association between common genetic variants and cancer risk, limiting the attention to single nucleotide polymorphisms (SNPs) of the NBN gene and providing the various odd ratios (ORs). In this respect, the NBN protein, together with MRE11 and RAD50, is part of the MRN complex which is a central player in the very early steps of sensing and processing of DNA double-strand breaks (DSBs), in telomere maintenance, in cell cycle control, and in genomic integrity in general. So far, many papers were devoted to ascertain possible association between common synonymous and non-synonymous NBN gene polymorphisms and increased cancer risk. However, the results still remain inconsistent and inconclusive also in meta-analysis studies for the most investigated E185Q NBN miscoding variant.

Is a single-site laparoendoscopic approach a real surgical advancement for the management of small renal masses?

Nephron-sparing surgery (NSS) ensures equivalent oncological results while improving overall survival compared with radical nephrectomy when applied to the treatment of small renal masses, moreover warm ischemia is associated with a risk of acute renal failure and advanced chronic kidney disease (CKD). Laparoendoscopic single-site (LESS) unclamp NSS is the next step forward in the management of small renal masses. From 2009 to 2013 we have treated 23 patients with small renal masses ( < 4 cm) amenable to the LESS approach using unclamp LESS NSS. In 20 cases we were able to complete the operation using LESS, in 3 cases conversion to standard laparoscopy was required. Pathologic examination revealed 16 cases of clear-cell renal cell carcinoma (RCC), 4 cases of renal cysts, 2 oncocytomas, and 1 angiomyolipoma. We did not find any significant variation in renal function or any case of tumor recurrence, and the majority of the patients were very satisfied of the cosmetic results. LESS unclamp partial nephrectomy is a safe and feasible procedure, oncological outcomes are similar to standard laparoscopy, there is an advantage with respect to renal function and cosmesis, although the procedure is more technically demanding compared with standard laparoscopy.

Clinical effects and economical impact of dutasteride and finasteride therapy in Italian men with LUTS.

OBJECTIVES: To investigate differences in the risk of benign prostatic hyperplasia (BPH)- related hospitalization, for surgical and non-surgical reasons, and of new prostate cancer (PCa) diagnosis between patients under dutasteride or finasteride treatment. MATERIAL AND METHODS: A retrospective cohort study was conducted using data from record-linkage of administrative databases. Men aged ≥ 40 years old who had received a prescription for at least 10 boxes/year (index years: 2004-06) were included. The association of the outcomes was assessed using a multiple Cox proportional hazard model. Propensity score matched analysis and a 5-to-1, greedy 1:1 matching algorithm were performed. The budget impact analysis of dutasteride vs finasteride in BPH-treated patient was performed. RESULTS: From an initial cohort of about 1.5 million of Italian men, 19620 were selected. The overall hospitalization for BPH-non surgical reasons, for BPH-related surgery and for new detection of PCa incidence rates (IRs) were 8.20 (95% CI, 7.62-8.23), 18.0 (95% CI, 17.12-18.93) and 8.62 (95% CI, 8.03-9.26) per 1000 person-years, respectively. The multivariate analysis after the propensity score-matching showed that dutasteride was associated with an independent reduced likelihood of hospitalization for BPH-related surgery (HR 0.82; 95% CI 0.73-0.93; p = 0.0025) and of newly detected PCa (HR: 0.76,95% CI, 0.65-0.85; p = 0.0116). The IR for BPH-non surgical reasons was 8.07 (95% CI, 7.10-9.17) and 9.25 (95% CI, 8.19-10.44) per 1000 person-years, respectively. The IR for BPH-related surgery was 18.28 (95% CI, 17.17-20.32) and 21.28 (95% CI, 19.24-23.06) per 1000 person-years among patients under dutasteride compared with those under finasteride, respectively. For new-onset PCa, the IR was 8.01 (95% CI, 7.07-9.08) and 9.38 (95% CI, 8.32-10.58) per 1000 person-years The pharmacoeconomical evaluation showed that the net budget impact of the use of dutasteride vs. finasteride in 1000 BPH-treated patient for 1 year induces a saving of 3933 €. CONCLUSIONS: The clinical effects of dutasteride and finasteride are slightly different. The likelihood of hospitalization for BPH-related surgery and of newly detected PCa seems to be in favor of dutasteride. The budget impact analyses showed a slightly benefit for dutasteride. Comparative prospective studies are necessary to confirm these results.

Drug repositioning strategy for the identification of novel telomere-damaging agents: A role for NAMPT inhibitors.

Drug repositioning strategy represents a valid tool to accelerate the pharmacological development through the identification of new applications for already existing compounds. In this view, we aimed at discovering molecules able to trigger telomere-localized DNA damage and tumor cell death. By applying an automated high-content spinning-disk microscopy, we performed a screening aimed at identifying, on a library of 527 drugs, molecules able to negatively affect the expression of TRF2, a key protein in telomere maintenance. FK866, resulting from the screening as the best candidate hit, was then validated at biochemical and molecular levels and the mechanism underlying its activity in telomere deprotection was elucidated both in vitro and in vivo. The results of this study allow us to discover a novel role of FK866 in promoting, through the production of reactive oxygen species, telomere loss and deprotection, two events leading to an accumulation of DNA damage and tumor cell death. The ability of FK866 to induce telomere damage and apoptosis was also demonstrated in advanced preclinical models evidencing the antitumoral activity of FK866 in triple-negative breast cancer-a particularly aggressive breast cancer subtype still orphan of targeted therapies and characterized by high expression levels of both NAMPT and TRF2. Overall, our findings pave the way to the development of novel anticancer strategies to counteract triple-negative breast cancer, based on the use of telomere deprotecting agents, including NAMPT inhibitors, that would rapidly progress from bench to bedside.

Proton Therapy, Magnetic Nanoparticles and Hyperthermia as Combined Treatment for Pancreatic BxPC3 Tumor Cells.

We present an investigation of the effects on BxPC3 pancreatic cancer cells of proton therapy combined with hyperthermia, assisted by magnetic fluid hyperthermia performed with the use of magnetic nanoparticles. The cells' response to the combined treatment has been evaluated by means of the clonogenic survival assay and the estimation of DNA Double Strand Breaks (DSBs). The Reactive Oxygen Species (ROS) production, the tumor cell invasion and the cell cycle variations have also been studied. The experimental results have shown that the combination of proton therapy, MNPs administration and hyperthermia gives a clonogenic survival that is much smaller than the single irradiation treatment at all doses, thus suggesting a new effective combined therapy for the pancreatic tumor. Importantly, the effect of the therapies used here is synergistic. Moreover, after proton irradiation, the hyperthermia treatment was able to increase the number of DSBs, even though just at 6 h after the treatment. Noticeably, the magnetic nanoparticles' presence induces radiosensitization effects, and hyperthermia increases the production of ROS, which contributes to cytotoxic cellular effects and to a wide variety of lesions including DNA damage. The present study indicates a new way for clinical translation of combined therapies, also in the vision of an increasing number of hospitals that will use the proton therapy technique in the near future for different kinds of radio-resistant cancers.